Massively parallel sequencing is unlocking the potential of environmental trace evidence.

Massively parallel sequencing (MPS) has revolutionised the field of genomics enabling substantial advances in human DNA profiling. Further, the advent of MPS now allows biological signatures to be obtained from complex DNA mixtures and trace amounts of low biomass samples. Environmental samples serve as ideal forms of contact trace evidence as detection at a scene can establish a link between a suspect, location and victim. Many studies have applied MPS technology to characterise the biodiversity within high biomass environmental samples (such as soil and water) to address questions related to ecology, conservation, climate change and human health. However, translation of these tools to forensic science remains in its infancy, due in part to the merging of traditional forensic ecology practices with unfamiliar DNA technologies and complex datasets. In addition, people and objects also carry low biomass environmental signals which have recently been shown to reflect a specific individual or location. The sensitivity, and reducing cost, of MPS is now unlocking the power of both high and low biomass environmental DNA (eDNA) samples as useful sources of genetic information in forensic science. This paper discusses the potential of eDNA to forensic science by reviewing the most explored applications that are leading the integration of this technology into the field. We introduce novel areas of forensic ecology that could also benefit from these tools with a focus on linking a suspect to a scene or establishing provenance of an unknown sample and discuss the current limitations and validation recommendations to achieve translation of eDNA into casework.

Ancestry and phenotype predictions from touch DNA using massively parallel sequencing.

Direct PCR can be used to successfully generate full STR profiles from DNA present on the surface of objects. STR profiles are only of use in cases where a potential donor profile is available for comparison, and DNA is of sufficient DNA quality and quantity to generate a reliable profile. Often, no donor information is available and only trace DNA is present on items. As a result, alternative techniques are required to generate genetic data that can provide investigative leads. Massively parallel sequencing (MPS) offers the ability to detect trace levels of DNA and improve DNA analysis success from touched items. Here, we present the first application of direct PCR coupled with MPS to generate forensic intelligence SNP data from latent DNA. The panels assessed are (1) the HIrisplex System that targets 24 SNPs to simultaneously predict hair and eye, and (2) the Precision ID Ancestry Panel that targets 165 autosomal SNPs indicative of biogeographic ancestry. For each panel, we analysed 60 touched samples across five individuals and four substrates (glass slide, fuse, zip-lock bag and wire) using Ion AmpliSeq Library Preparation Kit on the automated Ion Chef System and Ion Torrent PGM. We examine the SNP recovery, concordance with reference samples and the genotype reproducibility from different substrates and donors. The results demonstrate the application of this approach for obtaining informative genetic from trace amounts of DNA.

Forensic validation of a SNP and INDEL panel for individualisation of timber from bigleaf maple (Acer macrophyllum Pursch).

Illegal logging is one of the largest illicit trades in the world, with high profits and generally low risks of detection and prosecution. Timber identification presents problems for law enforcement as traditionally used forensic methods such as wood anatomy and dendrochronology are often unable to confidently match wood evidence to the remains of illegally felled trees. Here we have developed and validated a set of genetic markers for individualisation in bigleaf maple (Acer macrophyllum), a high value timber species often felled illegally in the USA. Using 128 single nucleotide polymorphisms and three insertion/deletion markers developed through massively parallel sequencing, 394 individuals were genotyped on the MassARRAY® iPLEX™ platform (Agena Bio-science™, San Diego, USA) to produce a population reference database for the species. We demonstrate that the resulting DNA assay is reliable, species specific, effective at low DNA concentrations (<1 ng/µL) and suitable for application to timber samples. The PID for the most common profile, calculated using an overall dataset level FST-correction factor, was 1.785 × 10-25 and PID-SIB across all individuals (treated as a single population) was 2.496 × 10-22. The further development of forensic identification assays for timber species has the potential to deliver robust tools for improved detection and prosecution of illegal logging crimes as well as for the verification of legality in reputable supply chains.

Enhancement of fingermarks and visualizing DNA.

A novel method for detection and visualization of latent DNA using Diamond™ Nucleic Acid Dye (DD) staining has been developed. Applying DD to an object has the real potential to visualize DNA on a substrate from which a DNA profile can be generated, but it is important to determine whether this staining will adversely affect other forensic investigational techniques and vice versa. The aim of this study was to examine the interactions between staining a fingermark to detect DNA and then generate a DNA profile in combination with several standard latent fingermark enhancement methods. Six common fingerprint enhancements processes were chosen; (1) black powder, (2) black magnetic powder, (3) red magnetic powder, (4) white powder, (5) aluminum powder and (6) cyanoacrylate fuming. For all six methods, mark enhancement was carried out before DD staining and vice versa. DD is effective in detection of DNA in the presence of both aluminum and white finger mark powders and DD does not compromise the subsequent detection of ridge patterns if DD is applied first. Whilst magnetic powders could be used to successfully enhance latent fingermarks even after DD had been applied to them, latent DNA could not be observed in the marks irrespective of whether magnetic powder was applied before or after DD treatment. Magnetic powders did not adversely affect the profiling of DNA present in the marks. The application of DD to fingermarks did not adversely affect the enhancement of fingermarks using cyanoacrylate fuming. Whilst fluorescent particles resembling cells stained with DD were observed in marks either post-treated or pre-treated with cyanoacrylate vapor, DNA amplification and profiling was not successful. While it may be important in particular investigations to collect DNA profiles from latent fingermarks with continuous ridges and clear minutiae, the main utility of the technique described here would be in relation to investigations where enhancement has resulted in only partial or smudged marks. The results presented here indicate that if it is desirable to visualize latent DNA on an object but it is also planned to treat the object with cyanoacrylate vapor or magnetic powders then it is important to apply DD first, record the location of DNA and then apply the mark enhancement technique. For aluminum and white powder mark treatments such precautions are not important.

Visualising latent DNA on swabs.

Collection for touch DNA either at scenes or on items submitted to a forensic laboratory is based on assumptions as to where a person made direct contact. In many instances a swab may be applied to an area where no contact has been made. Many swabs may therefore be submitted for DNA profiling on which no DNA is present, resulting in the loss of both time and resources by analysing such swabs. This study has developed a simple, fast, DNA-staining and fluorescence microscopy-based screening method for swabs to indicate if there is any DNA from which to generate a profile. Ten different types of swabs were tested covering the major types used (foam, cotton and nylon). Each swab was treated by: no addition of dye or DNA, addition of dye only, addition of known DNA and addition of dye and DNA. The stain used was Diamond™ Nucleic Acid Dye (DD) and fluorescence microscopy was achieved with a digital microscope equipped with a blue LED light source (480nm) for excitation and an emission filter of 510nm. Two types of samples were tested, either buccal swabs or swabs collected from areas touched by volunteers and all analyses were performed in triplicate. The samples were collected and retained at room temperature with time intervals of 0 day, 7 days, 14 days, 21 days, and 28 days before detection using DD staining and fluorescence microscopy. Seven of the swab types used were found to be unsuitable due to the lack of any difference in the fluorescence detected when no DNA, or only the dye, or a combination of DNA and dye were added. Three swab types (black cotton swab, Ultrafine dental applicator, and Cylinder dental applicator) were found to be much more effective for collection of DNA. Further, stained cellular material retained its fluorescence for up to 4 weeks and swabs containing cellular material that had been stored for four weeks could be stained and visualised. Additionally, DD did not affect DNA profiling. This screening method has the potential to be a routine step in a forensic laboratory to save costs of processing samples where swabs are devoid of any DNA. This technique is rapid, easy, cheap, non-destructive and safe.

"Bottom-up" in situ proteomic differentiation of human and non-human haemoglobins for forensic purposes by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry.

RATIONALE: The detection and identification of human blood on crime-related items are of particular relevance to many investigations because shed blood can provide evidence of violent contact between individuals. However, for any detection and identification technique, specificity is a critical performance characteristic to assess; that is, whether the technique has the capability to differentiate between human blood (which usually is of relevance to a criminal investigation) and non-human blood (which usually would not be associated with a crime but may be detected incidentally). METHODS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approaches using "top-down" (detection of intact proteins) and "bottom-up" (detection of tryptic peptide markers) were used to detect and identify haemoglobin in blood from humans and from a range of Australian native mammals; the technique could be carried out directly on blood stains without the need to extract proteins (i.e., in situ measurement). Imaging of haemoglobin was achieved in bloodied fingermarks, including those that had been enhanced using two "industry standard" fingermark enhancement processes. RESULTS: Differentiation of intact haemoglobin proteins in human and non-human blood using "top-down" MALDI-TOF-MS was difficult. However, in situ "bottom-up" approaches using tandem mass spectrometry (MS/MS) and de novo sequencing of tryptic digest peptides allowed unambiguous differentiation. Imaging mass spectrometry of human haemoglobin, even when it was mixed with animal blood, was achieved in bloodied fingermarks that had been enhanced using two common processes (staining with Amido Black or dusted with magnetic powder) and "lifted" using adhesive tape. CONCLUSIONS: The MALDI-TOF-MS-based in situ "bottom-up" proteomic methodology described here shows great promise for the detection of human blood and even imaging of blood in bloodied fingermarks. The approach is sensitive, can differentiate between human blood and that from many animals (including several Australian native animals), and can be implemented after traditional crime scene fingermark enhancement techniques have been carried out.

Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker (AIM) panels: Results of a collaborative EDNAP exercise.

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.

ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations.

The use of non-human DNA typing in forensic science investigations, and specifically that from animal DNA, is ever increasing. The term animal DNA in this document refers to animal species encountered in a forensic science examination but does not include human DNA. Non-human DNA may either be: the trade and possession of a species, or products derived from a species, which is contrary to legislation; as evidence where the crime is against a person or property; instances of animal cruelty; or where the animal is the offender. The first instance is addressed by determining the species present, and the other scenarios can often be addressed by assigning a DNA sample to a particular individual organism. Currently there is little standardization of methodologies used in the forensic analysis of animal DNA or in reporting styles. The recommendations in this document relate specifically to animal DNA that is integral to a forensic science investigation and are not relevant to the breeding of animals for commercial purposes. This DNA commission was formed out of discussions at the International Society for Forensic Genetics 23rd Congress in Buenos Aires to outline recommendations on the use of non-human DNA in a forensic science investigation. Due to the scope of non-human DNA typing that is possible, the remit of this commission is confined to animal DNA typing only.

Forensic Science in Support of Wildlife Conservation Efforts - Genetic Approaches (Global Trends).

Wildlife forensic science is a relatively recent development to meet the increasing need of the criminal justice system where there are investigations in alleged transgressions of either international or national legislation. This application of science draws on conservation genetics and forensic geneticists from mainstream forensic science. This review is a broad overview of the history of forensic wildlife science and some of the recent developments in forensic wildlife genetics with the application of DNA developments to nonhuman samples encountered in a forensic science investigation. The review will move from methods to look at the entire genome, when there is no previous knowledge of the species studied, through methods of species identification, using DNA to determine a possible geographic origin, through to assigning samples to a particular individual or a close genetic relative of this individual. The transfer of research methods into the criminal justice system for the investigation of wildlife crimes has been largely successful as is illustrated in the review. The review concludes with comments on the need for standardization and regulation in wildlife forensic science.

Application of Mitochondrial DNA Technologies in Wildlife Investigation - Species Identification.

Forensic science laboratories are increasingly requested to investigate crimes involving wildlife and to examine nonhuman biological tissue. The illegal trade in endangered species has led to a need to use methods, accepted by the forensic community and acceptable to a court of law, that will identify species that are present from seized samples and to determine whether the ownership of the samples contravenes any legislation. The use of genetic loci on the mitochondrial genome has become standard with the cytochrome b gene being the best described and utilized of the mitochondrial genes. The amplification of part or all of the gene and comparison to stored DNA sequences held on DNA databases such as EMBL (by the European Molecular Biology Laboratory) or GenBank (by the National Institutes of Health) can lead to the identification of the species. Examination of single nucleotide polymorphisms is possible in the case of highly degraded samples. The testing methods available have led to the successful prosecution of traders in endangered or protected species.

STR data for the SGM Plus loci from three Indonesian populations.

Allele frequencies for the 10 STRs included in the AmpFLSTR SGM Plus (Applied Biosytems) amplification kit were obtained from three populations in the Indonesian archipelago. Here, 173 unrelated Indonesian individuals were sampled, of which 44 were from the Island of Sulawesi, 44 from Sumatra and 85 from Java.

Cytochrome b gene for species identification of the conservation animals.

A partial DNA sequence of cytochrome b gene was used to identify the remains of endangered animals and species endemic to Taiwan. The conservation of animals species included in this study were: the formosan gem-faced civets, leopard cats, tigers, clouded leopards, lion, formosan muntjacs, formosan sika deers, formosan sambars, formosan serows, water buffalo, formosan pangolins and formosan macaques. The control species used included domestic cats, domestic dogs, domestic sheeps, domestic cattles, domestic pigs and humans. Heteroplasmy was detected in the formosan macaque, domestic pig and domestic cats. The frequencies of heteroplasmy in these animals were about 0.25% (1 in 402bp). Sequences were aligned by Pileup program of GCG computer package, and the phylogenetic tree was constructed by the neighbor-joining method. The results of sequence comparison showed that the percentage range of sequence diversity in the same species was from 0.25 to 2.74%, and that between the different species was from 5.97 to 34.83%. The results of phylogenetic analysis showed that the genetic distance between the different species was from 6.33 to 40.59. Animals of the same species, both the endangered animal species and domestic animals, were clustered together in the neighbor-joining tree. Three unknown samples of animal remains were identified by this system. The partial sequence of cytochrome b gene adopted in this study proved to be usable for animal identification.

STR data for the GenePrint PowerPlex 1.2 system loci from three United Arab Emirates populations.

Allele frequencies for the eight STRs included in the GenePrint PowerPlex 1.2 kit were obtained from three population groups that are resident in the United Arab Emirates (UAE); 228 unrelated UAE individuals, 194 unrelated Indian individuals and 197 unrelated Pakistani individuals were analysed.

A novel nomenclature for the hypervariable short tandem repeat APOAI1.

A novel nomenclature for the hypervariable microsatellite DNA, APOAI1 locus, is proposed. The complex nature of the repeat unit in this locus results in alleles separated by a single base. Polymerase chain reaction (PCR) products amplified from this locus were separated by single-strand conformation polymorphism (SSCP) electrophoresis. All the single-stranded DNA bands on the SSCP gel were removed from the gel and a second amplification performed. Homozygous DNA fragments amplified from single-stranded DNA were sequenced. From the 100 individuals studied, 30 alleles and 73 genotypes were found. A system of nomenclature for the APOAI1 locus is provided that is logical and in line with previous models. Using the primers described, the locus can be amplified and alleles designated on the basis of size. This system of nomenclature will assist in the exchange of data between laboratories for this locus.

Sequence polymorphism of mitochondrial D-loop DNA in the Taiwanese Han population.

In order to demonstrate the sequence diversity of mitochondrial D-loop DNA in the Taiwanese Han population, we established a database of 155 unrelated individuals. For each individual, the complete 980bp DNA region from the 5' end of HVI to 3' end of HVII segment was sequenced. In these 155 sequence data, 149 different haplotypes were observed, amongst these haplotypes, 144 were unique, 4 were found in 2 individuals and 1 was found in 3 individuals. When compare to the Anderson sequence, 144 transitions, 24 transversions, 5 insertions and 5 deletions were found. Eight positions exhibited more than one polymorphic sequence, six exhibited two variants while two exhibited three variants. Over the 1024bp that was analysed, pairwise differences between the sequences were 11.35+/-3.53bp. The sequence and nucleotide diversity were 0.9994 and 0.0116, respectively. The probability of two individuals randomly matching over the entire control region was 0.007. The diversity in the mitochondrial D-loop indicates the value of this locus for casework within Taiwan.

STR population data for 10 STR loci including the GenePrint PowerPlex 1.2 kit from El-Minia (Central Egypt).

Allele frequencies for 10 STRs including the GenePrint PowerPlex 1.2 loci and also D3S1358, HumvWA and HumFGA were obtained from a sample of unrelated individuals from El-Minia City.

13 STR loci frequency data from a Scottish population.

A total of 13 STR loci were typed from over 200 Caucasians from the Strathclyde region in the west of Scotland.

Identification of members of the genera Panaeolus and Psilocybe by a DNA test. A preliminary test for hallucinogenic fungi.

Abuse of hallucinogens produced by the fungal genera Psilocybe and Panaeolus are a growing problem. Five species from each of the two genera were examined in this preliminary research and a method that will unambiguously identify fungal samples as being of one of these two genera has been developed. The method uses genus specific DNA sequences within the Internal Transcribed Spacer of the ribosomal gene complex. Amplification of a common DNA product and a genus specific product results in two identifiable products, which facilitates the unambiguous identification of material from these two fungi to generic level.

Identification of hallucinogenic fungi from the genera Psilocybe and Panaeolus by amplified fragment length polymorphism.

Unambiguous identification of the hallucinogenic fungi of the genera Psilocybe and Panaeolus is required by national and international drug control legislation. We report on a DNA-based test using the technique of amplified fragment length polymorphism (AFLP). AFLP can differentiate species of the two genera Psilocybe and Panaeolus by using different primer sets. The identification of hallucinogenic fungi using a DNA-based test, which can be used in conjunction with morphological features, will assist in forensic investigations.